Small intestine inflammation in Roquin-mutant and Roquin-deficient mice

Abstract

Roquin, an E3 ubiquitin ligase that localizes to cytosolic RNA granules, is involved in regulating mRNA stability and translation. Mice that have a M199R mutation in the Roquin protein (referred to as sanroque or Roquin(san/san) mice) develop autoimmune pathologies, although the extent to which these occur in the intestinal mucosa has not been determined. Here, we demonstrate that Roquin(san/san) mice reproducibly develop intestinal inflammation in the small intestine but not the colon. Similarly, mice generated in our laboratory in which the Roquin gene was disrupted by insertion of a gene trap cassette (Roquin(gt/gt) mice) had small intestinal inflammation that mimicked that of Roquin(san/san) mice. MLN cells in Roquin(san/san) mice consisted of activated proliferating T cells, and had increased numbers of CD44(hi) CD62L(lo) KLRG1(+) short-lived effector cells. Proportionally more small intestinal intraepithelial lymphocytes in Roquin(san/san) mice expressed the ICOS T cell activation marker. Of particular interest, small intestinal lamina propria lymphocytes in Roquin(san/san) mice consisted of a high proportion of Gr-1(+) T cells that included IL-17A(+) cells and CD8(+) IFN-γ(+) cells. Extensive cytokine dysregulation resulting in both over-expression and under-expression of chemotactic cytokines occurred in the ileum of Roquin(san/san) mice, the region most prone to the development of inflammation. These findings demonstrate that chronic inflammation ensues in the intestine following Roquin alteration either as a consequence of protein mutation or gene disruption, and they have implications for understanding how small intestinal inflammation is perpetuated in Crohn's disease (CD). Due to the paucity of animal models of CD-like pathophysiology in the small intestine, and because the primary gene/protein defects of the Roquin animal systems used here are well-defined, it will be possible to further elucidate the underlying genetic and molecular mechanisms that drive the disease process.

Figure 1. Morphometric and intestinal histopathological features of Roquin^san/san mice.

(A) MS-PCR was used to identify homozygous Roquin^san/san mutant as demonstrated by the presence of a single 215 bp PCR product. Heterozygous Roquin^san/san mice had two PCR products. Roquin^san/san mice employed in the study were confirmed by MS-PCR. (B, C) Body weight of Roquin^san/san mice 15–55 weeks of age was significantly less than that of gender-matched normal animals. Mean ± SEM of 13 normal and 12 Roquin^san/san mice. (D) Roquin^san/san mice also had pronounced splenomegaly with follicular lymphoid hyperplasia, a characteristic of this animal model. Mean ± SEM of 7 normal and 12 Roquin^san/san mice ages 15–55 weeks. (E) Histopathological analysis of intestinal tissues revealed inflammation throughout the small intestine and cecum. Mean ± SEM of pathology scores of tissues from 23 Roquin^san/san mice and 8–10 normal mice ages 15–55 weeks. (F) Distribution of pathology scores for the ileum of Roquin^san/san mice as shown in panel E; note that only 2 of 23 Roquin^san/san mice had no inflammation or mucosal injury in the ileum. (G–I) Representative H&E stained tissue sections from the ileum of Roquin^san/san mice with pathology scores of 2, 3, and 4 that were characterized by villous atrophy and ulceration, crypt hyperplasia and increased intraepithelial mononuclear cell infiltrate. Original magnification, 200x. (J) Icos gene expression was elevated in the ileum of Roquin^san/san mice. Mean ± SEM of three replicate samples ages 16, 17, and 26 weeks.

Figure 2. Roquin^san/san mice have chronic hepatitis.

(A) Average pathology score of normal and Roquin^san/san mice. Mean ± SEM of 6 normal and 26 Roquin^san/san mice ages 15–55 weeks. (B, C) Representative H&E stained tissue sections of the liver from Roquin^san/san mice with pathology scores of 5. (C) Mononuclear cell infiltrates are seen distributed in the periportal area (PP) and around the central veins (CV) of the hepatic lobules. Focal areas of hepatocyte necrosis (arrows) with mononuclear cell infiltration is found in the mid-zonal and pericentral areas of the hepatic lobules. (D) A representative H&E stained liver section from a normal C57BL/6 mouse devoid of inflammation. Original magnifications, B and D, 100×; C, 200×.

Figure 3. Roquin^gt/gt gene trap mice develop extensive small intestine and liver inflammation.

(A) The location of the β-geo gene trap cassette and genotyping primers relative to the Roquin exons. (B) Roquin^gt/gt mice were screened using gene trap-specific primers. Mice homozygous for the gene trap had a single band of 800 bp; heterozygotes had two bands of 800 bp and 486 bp; wild type mice had a single 486 bp band. Animal ages 16–22 weeks. (C) The Roquin protein was undetectable in the spleen of homozygous (gt/gt) mice by western blotting; 1animal each. Animal ages 16–22 weeks. (D) Roquin gene expression in the intestine of homozygous (gt/gt) mice was reduced by ∼95%; 1animal each. Animal ages 16–22 weeks. Representative H&E stained tissue sections of the (E) liver, (F) duodenum, (G) jejunum, (H) ileum, and (I) cecum of a Roquin^gt/gt mouse exhibited chronic hepatitis and intestinal inflammation. Pathology scores of sections are described in the text. E–I, original magnifications, 200×.

Figure 4. MLN cells in Roquin^san/san mice have increased numbers of activated T cells, proliferating cells, and regulatory T cells.

MLN lymphocytes from (A) normal and (B) Roquin^san/san mice based on expression of OX40, ICOS, B220, FoxP3, and BrdU incorporation. FoxP3 CD25 expression was determined after gating onto CD4⁺ cells. Representative data from 2–3 normal and 2–4 Roquin^san/san mice.

Figure 5. MLN cells from Roquin^san/san mice are proliferative and have more SLECs.

Based on Ki67 staining, (panel B) a greater proportion of OX40⁺ cells and ICOS⁺ MLN cells were proliferating T cells compared to MLN cells from (panel A) normal mice. Representative staining from 1 normal and 2 Roquin^san/san mice. Similarly, there was a greater proportion of CD44^hi CD62L^lo KLRG1⁺ SLECs present in MLN cells of (panel D) Roquin^san/san mice compared to (panel C) normal mice. Representative data from 3 normal and 3 Roquin^san/san mice.

Figure 6. Characterization of siIELs in Roquin^san/san mice.

siIELs from Roquin^san/san mice have more (panel B) ICOS⁺ T cells, including a subset of CD44^hi ICOS⁺ cells compared to (panel A) normal animals. Interestingly, neither IL-17A nor IFN-γ were produced to any appreciable levels in either type of mouse (panels A and B). Representative data from 2 normal and 2 Roquin^san/san mice.

Figure 7. Unique phenotypic profile of siLPLs in Roquin^san/san mice.

siLPLs in Roquin^san/san mice differed fundamentally from those in normal mice in that Roquin^san/san mice had an unusually high proportion of GR-1⁺ T cells as determined by CD3 expression. The majority of those cells were CD44⁺ and they included a subset of IL-17A secreting cells. A subset of CD8⁺ Gr-1⁺ cells express IFN-γ (panel B compared to panel A). Representative data from 2–3 normal and Roquin^san/san mice.